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L Grossman

Publications and source records attributed to L Grossman.

At least 19 recordsLinked to original sources
PubMed1992

A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair.

The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.

Anthramycin↗
PubMed1992

Safety and immunogenicity of acellular pertussis vaccine combined with diphtheria and tetanus toxoids in 17- to 24-month-old children.

A double-blind, randomized, controlled trial comparing 4 lots of acellular pertussis-diphtheria tetanus toxoids vaccine (APDT) to whole cell DTP vaccine in 397 children was conducted at 7 clinical centers. Children were immunized at 17 to 24 months of age and sera were obtained pre- and postimmunization. Sera were analyzed for antibody to pertussis antigens (pertussis toxin, filamentous hemagglutinin, with a molecular weight of 69,000 (69k) outer membrane protein and agglutinogens) and to diphtheria and tetanus toxoids. Information concerning local reactions and systemic events was collected daily for 10 days postimmunization. The acellular vaccine produced significantly fewer local reactions than whole cell DTP. Parents reported that drowsiness or fretfulness occurred significantly less often in APDT vaccine recipients compared with whole cell DTP recipients. Fever greater than or equal to 38.3 degrees C occurred in 8% of APDT vaccine recipients and in 15% of whole cell DTP vaccine recipients (P = 0.06). The only significant difference in immune response to pertussis antigens between the two vaccines was for filamentous hemagglutinin (P less than 0.01) for which significantly higher antibody concentrations were found in the APDT vaccine group. We conclude that this APDT vaccine is safe and immunogenic when administered as a booster dose to 18-month-old children.

Antibodies, Bacterial↗
PubMed1991

Development and field-test validation of an assay for DNA repair in circulating human lymphocytes.

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.

Adult↗
PubMed1991

Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies.

The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro. In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins. The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction. The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA. Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function.

Adenosine Triphosphatases↗
PubMed1991

Deletion mutagenesis of the Escherichia coli UvrA protein localizes domains for DNA binding, damage recognition, and protein-protein interactions.

The UvrA protein is the DNA binding and damage recognition subunit of the damage-specific UvrABC endonuclease. In addition, it is an ATPase/GTPase, and the binding energy of ATP is linked to dimerization of the UvrA protein. Furthermore, the UvrA protein interacts with the UvrB protein to modulate its activities, both in solution and in association with DNA, where the UvrAB complex possesses a helicase activity. The domains of the UvrA protein that sponsor each of these activities were localized within the protein by studying the in vitro properties of a set of purified deletion mutants of the UvrA protein. A region located within the first 230 amino acids was found to contain the minimal region necessary for interactions with UvrB, the UvrA dimerization interface was localized to within the first 680 amino acids, and the DNA binding domain lies within the first 900 amino acids of the 940-amino acid UvrA protein. Two damage recognition domains were detected. The first domain, which coincides with the DNA binding region, is required to detect the damage. The second domain, located on or near the C-terminal 40 amino acids, stabilizes the protein-DNA complex when damage is encountered.

Adenosine Triphosphatases↗
PubMed1991

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.

Adenosine Triphosphatases↗
PubMed1991

Dimerization of Escherichia coli UvrA and its binding to undamaged and ultraviolet light damaged DNA.

The initial stages in the repair of damaged DNA by the Escherichia coli uvr system involve the recognition of damage by UvrA. We have examined in detail the binding of UvrA to DNA randomly damaged by ultraviolet light, undamaged DNA, and single-stranded DNA using nitrocellulose filter binding and gel mobility shift assays to arrive at the following model: UvrA dimers bind specifically to damaged DNA both in the presence and in the absence of ATP. The dimerization of UvrA is promoted by UvrA concentrations greater than 1 nM, the presence of ATP, or physiological temperatures, and the dimerization step dominates the temperature dependence of UvrA binding to DNA damaged by ultraviolet light. The apparent association constant for specific binding is dependent on the concentration of UvrA due to coupled dimerization, aggregation, and nonspecific binding reactions. At 1 nM UvrA, either with or without ATP, Kuv approximately 10(9) M-1. The binding of UvrA to undamaged DNA is 10(3)-10(4)-fold weaker than the damage-specific binding. Both the strength of damage-specific binding and the discrimination between damaged and undamaged sites are affected by the salt concentration. The kinetics of association and dissociation reactions indicate that the primary effects of ATP are on the extent of UvrA dimerization rather than on the properties of the UvrA-uvDNA complex. The complexity of the interaction of UvrA, ATP, and DNA is indicated by the opposing effects of ATP binding and hydrolysis on UvrA dimerization.

Adenosine Triphosphatases↗
PubMed1991

ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB.

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAB complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. Our results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed.

Adenosine Triphosphate↗
PubMed1990

The role of Escherichia coli UvrB in nucleotide excision repair.

The role of UvrB in determining the nucleotide dependence of Escherichia coli excision repair has been investigated. The mutation of lysine 45 in the ATPase motif of UvrB to alanine leads to an acute defect in ATP hydrolysis and failure to support incision of UV-damaged DNA. This ATP hydrolysis activity is not required for interaction of UvrB with UvrA in solution, or for formation of a damage-independent nucleoprotein complex in the presence of UvrA and nucleotide. This UvrB mutant fails, however, to support damage-specific nucleoprotein complex formation, and does not participate in a UvrA-UvrB-dependent helicase-like activity. We conclude from these results that mutation at lysine 45 in the ATPase motif of UvrB specifically inhibits a key step in nucleotide excision repair involving the UvrB ATPase-dependent translocation of nucleoprotein complexes from undamaged to damaged DNA sites.

Adenosine Triphosphatases↗
PubMed1990

Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.

Cell-Free System↗
PubMed1990

The Pediatric Early Elementary Examination: sensitivity and specificity.

Evaluated the sensitivity and specificity of the Pediatric Early Elementary Examination (PEEX), a test designed to identify children at risk for learning problems. Using a sample of 299 seven- to nine-year-old "disadvantaged" children, the number of "areas of concern" identified by the PEEX and a school record indicating whether a child was receiving special educational services were used to compute test sensitivity and specificity. For two areas of concern, the sensitivity was 76.9% and specificity was 59.9%. The findings indicate that the PEEX correctly identified only 64.3% of children receiving special educational services. The test has a high rate of false positives while still failing to identify 6-7.5% of the children receiving special education services. Other issues relating to the utility of the PEEX are discussed.

Child↗
PubMed1989

ATPase activity of the UvrA and UvrAB protein complexes of the Escherichia coli UvrABC endonuclease.

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.

Adenosine Triphosphatases↗
PubMed1989

Characterization of the helicase activity of the Escherichia coli UvrAB protein complex.

The requirement for nucleotide hydrolysis in the DNA repair mechanism of the Escherichia coli UvrABC protein complex has been analyzed. The DNA-activated UvrAB ATPase activity is part of a helicase activity exhibited by the UvrAB protein complex. The helicase acts only on short duplexes and, therefore, is unlike other helicases such as those involved in DNA replication that unwind long duplexes. The strand displacement activity occurs in the 5'----3' direction and requires either ATP or dATP. The helicase activity is inhibited by UV photoproducts. The absence of this activity in a complex formed with proteolyzed UvrB (UvrB*), a complex also deficient in the endonuclease activity, suggests that this activity is important in the repair mechanism. The UvrAB protein complex may remain bound to a damaged site and by coupling the energy derived from ATP hydrolysis, alter the DNA conformation around the damage site to one that is permissive for endonucleolytic events. The conformational changes may take the form of DNA unwinding.

DNA Helicases↗
PubMed1989

Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity.

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

Amino Acid Sequence↗
PubMed1989

Dynamics of the Escherichia coli nucleotide excision repair system.

Ultraviolet light induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. At the highest level of complexity Escherichia coli (E. coli) has a uvr DNA repair system comprising the UvrA, UvrB and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding, is associated with localized topological unwinding of DNA. This protein complex can catalyze an ATP-dependent 5'----3' directed strand displacement of D-loop DNA or short single strands annealed to a single stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled "excision resynthesis" step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress induced protease which also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.

DNA Damage↗